Proliferative cells, such as fibroblasts, are well known to have a very "plastic" cytoskeleton. In contrast, striated muscle cells are known to have a rigid cytoskeleton. Differentiated vascular smooth muscle cells (dVSMCs), based on limited existing literature and our preliminary data, appear to function somewhere in between these two cell types. The long-term goal of this project is to determine to what degree and by what mechanisms the "non-muscle" cytoskeleton modulates smooth muscle contractility during agonist-, hydrodynamic and disease-induced remodeling of its structure. The Specific Aims over the next 5 years are: 1. To test the hypothesis, based on preliminary data, that actin filaments remodel during agonist stimulation and stretch in dVSMCs and to determine whether this alters contractility. 2. To test the hypothesis that specific nucleation/elongation and stabilizing proteins are involved in cytoskeletal remodeling of dSMC, focusing first on profilin, N-WASP, and VASP (nucleating/elongation), and CaD, Tm, and CaP (stabilizing). We will also test the hypothesis that the distribution of smooth muscle putative filament stabilizer proteins, CaD, Tm, and CaP, play a role in defining the localizationof the less labile actin filaments. 3. To test the hypothesis that remodeling of the dense plaques and/or dense bodies and/or their attachements to actin filaments occurs in dVSMCs, and, that the non-receptor tyrosine kinases FAK and Src regulate cytoskeleton/plaque remodeling in dVSMC. Techniques will include differential centrifugation, and in vivo labeling to monitor F and G actin populations, 2-color immunoblot, mass spectrometry, in-cell photoaffinity cross-linking and FRET, cell permeant decoy peptides, dominant negative protein fragments, antisense technology and standard cellular, biochemical and molecular methodologies. The results from this project will further our understanding of the, yet unclear mechanisms offeree maintenance in the dVSMC and will increase our fundamental understanding of the role of the nonmuscle cytoskeleton in the control of vascular function. LAY SUMMARY: The limitations of current drugs to treat cardiovascular disease is due, in part, to our limited understanding of how the muscle cells of blood vessels function. This project will expand our understanding of the VSMC and has the potential of identifying entirely novel targets for drug development.